The disclosures of all publications mentioned below, whether patent or non-patent documents, are incorporated herein by reference.
Pre-eclampsia is a pregnancy-related disease characterised by hypertension, proteinuria and oedema. It is responsible for around 12% of the world's annual 514,000 maternal deaths [Reference 1]. Aside from maternal and fetal death, the condition can also result in intra-uterine growth restriction, seizures (eclampsia), renal or liver failure, and placental abruption. Despite much investigation, the pathological processes underlying this disease are still largely undiscovered. Recent investigation had focussed on defective placental implantation as an important aetiological factor, with the resulting release of placentally derived circulating factors, which cause endothelial dysfunction [References 2, 3, 4]. At the microvascular level, there is a state of vasoconstriction from smooth muscle contraction, increased vascular permeability and anti-angiogenesis [Reference 5], which correspond to the clinical findings of high blood pressure, oedema and a characteristically small placenta at delivery of the baby.
The vascular endothelial growth factor (VEGF) family is thought to be one of the important molecular systems involved in the pathogenesis of pre-eclampsia. Conventional VEGF, also known as VEGF-A, is made up of 6 different isoforms formed from alternative exon splicing resulting in proteins of varying amino acid length, termed VEGFxxx. VEGF165 is the most common isoform of VEGFxxx, and consists of 165 amino acids. VEGF165 acts via its receptor VEGFR-2 to increase vascular permeability, vasodilatation and angiogenesis [Reference 6]. Endogenous alternative splicing of the VEGF receptor results in soluble VEGFR-1 (also known as soluble fms-like tyrosine kinase 1 or sFlt-1), which binds to VEGF and inhibits its function [Reference 6]. High levels of sFlt-1 have been documented in pre-eclampsia [Reference 7].
VEGF levels in pre-eclampsia have been measured by a number of techniques, with conflicting results according to the technique used. When measured by commercial sandwich Enzyme Linked ImmunoSorbent Assays (ELISAs)—which has been proposed to measure only the free, unbound forms of VEGF-levels appear to be reduced in pre-eclampsia [References 8, 9]. When measured by radioimmunoassay (RIA) or competitive enzyme immuno assay (cEIA), VEGF levels are shown to substantially increase. This discrepancy has been proposed to be due to these latter two methods not being affected by circulating binding proteins [References 10, 11].
In 2002, an alternative family of VEGF-A isoforms were identified, termed VEGFxxxb. These are the same size as conventional VEGF-A but are alternatively spliced in exon 8 [Reference 12]. This alternative splice site selection results in an alternate 6 amino acid C terminus, which affects the property of the isoforms. VEGF165b is the most widely studied of these isoforms. VEGF165b has been shown to inhibit the effects of VEGF165 by binding to its principal receptor VEGFR-2 and preventing it from exerting its physiological effects such as endothelial cell proliferation and migration. VEGF165b also binds to and activates Flt-1 (VEGFR-1), resulting in a transient increase in capillary hydraulic conductivity but no sustained increase in permeability, in contrast with VEGF165 [Reference 13].
WO03/012105 describes the use of VEGF165b inhibitors, for example anti-VEGF165b antibodies, to treat pre-eclampsia associated with a lack of VEGF165-mediated vasodilation. The rationale underlying this treatment is explained at page 23, line 21 to page 24, line 12. However, Bates et al, [Reference 29] report that pre-eclamptic placentae at term have significantly down-regulated levels of VEGFxxxb, implying a different mechanism than merely excess VEGF165b expression. Evidence is presented to indicate a significant uncoupling of the splicing regulation of the VEGF isoforms in late pre-eclampsia. It is theorised that such dysregulation of mRNA splicing in VEGF gene expression in pre-eclampsia may be linked to apparent dysregulation of mRNA splicing in expression of the VEGFR (VEGF receptor) gene, also observed in human pre-eclampsia.
We have now surprisingly found that a delay in the up-regulation of VEGFxxxb, for example VEGF165b, in the pregnant maternal plasma from the non-pregnant level to a higher concentration is an early marker indicative of risk of pre-eclampsia later in the pregnancy. This longitudinal parameter can therefore be used as the basis for a predictive assay for risk of pre-eclampsia.
It follows that clinical intervention in about the first trimester of the pregnancy to restore maternal plasma concentration of VEGFxxxb, for example VEGF165b, to or towards normal levels will provide a valuable treatment for reducing a risk of development of pre-eclampsia later in the pregnancy or for delaying onset of pre-eclampsia, thereby improving the prospects of fetal and maternal survival. Aspects of this study are described in Bills et al, Clinical Science (2009) 116, pages 265-272 (“Failure to Up-Regulate VEGF165b in Maternal Plasma is a First Trimester Predictive Marker for Pre-Eclampsia”). To the extent that any part of that publication or any related publication prior to the filing of this patent application would otherwise be prior art against the invention under the relevant law, we claim the benefit of any grace period provided by the relevant law.